目的：本研究通过观察经地塞米松处理前后培养的人小梁细胞的变化,探讨激素性青光眼房水排出阻力增加的机制。方法：将人类小梁细胞培养至接近体内的高分化状态，用地塞米松处理后,观察细胞形态的变化，并检测以下蛋白的分布和表达：(1)myocilin/TIGR蛋白；(2)纤连蛋白；(3)肌动蛋白交联网的形成；(4)血清淀粉样物质A蛋白。结果：人小梁细胞经地塞米松处理后(1)胞体变大，排列不规则，边界模糊，呈“融合”状；(2)细胞内外myocilin/TIGR蛋白表达均明显增加，其胞外表达与纤连蛋白位置一致,提示相互作用；(3)纤连蛋白表达增加；(4)CLANs形成，细胞间连接增强；(5)血清淀粉样物质A蛋白表达增加。结论：人类小梁细胞经地塞米松处理后形态发生变化，可能与应力纤维变化及myocilin蛋白在细胞内积聚有关。细胞间边界不清，与细胞外基质蛋白过度表达、沉积有关。小梁细胞外基质沉积以及异常的细胞间连接等改变与房水排出阻力增加的病理过程有关。
Abstract： Objective To study the molecular mechanism of pathologic states related to cardiopulmonary bypass (CPB) and screen the differential proteins from the serum before and after CPB in the open heart surgery patients. Methods By the twodimensional gel electrophoresis (2DE), we took the blood samples from each of the sixteen open heart surgery patients 30 minutes before CPB, 1 hour after CPB, and 24 hours after CPB. The protein spots were analyzed by the PDQuest image analysis software and the differential protein spots were identified by matrixassisted laser desorption/ionizationtime of flightmass spectrometry (MALDITOF-MS). Then, enzymelinked immunosorbent assay (ELISA) was used to determine the expression level of serum amyloid A protein (SAA) in the serum of healthy people and the enrolled patients before and after CPB. Results Through 2DE in combination with massspectrometry, 7 proteins altered in expression were identified, including SAA, haptoglobin (HPT), leucinerich alpha2-glycoprotein (A2GL), hemoglobin subunit beta (HBB), serine/threonineprotein phosphatase 2A -regulatory subunit B″ subunit gamma (P2R3C), transthyretin (TTHY), and T-complex protein 11-like protein2 (T11L2). ELISA analysis showed that SAA levels in healthy people and the open heart surgery patients 30 minutes before CPB were not statistically different (t=-1.955, P=0.056), while the SAA level rose from 54.47±48.32 μg/ml 30 min before CPB to 1 017.78±189.92 μg/ml 24 hours after CPB in the serum of open heart surgery patients． Conclusion The results of this pilot study illustrate that SAA, HPT, A2GL, HBB, P2R3C, TTHY and T11L2 may be the molecule markers of pathologic state related to CPB. Acute phase reaction happens intensively after CPB in human body.
Objective To generate eukaryotic expression vector of pcDNA3.1-β-site amyloid precursor protein cleaving enzyme (BACE) and obtain its transient expression in COS-7 cells. Methods A 1.5 kb cDNA fragment was amplified from the total RNA of the human neuroblastoma cells by the RT-PCR method and was cloned into the plasmid pcDNA3.1. The vector was identified by the double digestion with restriction enzymes BamHI and XhoI and was sequenced by the Sanger-dideoxy-mediated chain termination. The expression of the BACE gene was detected by immunocytochemistry. Results The results showed that the cDNA fragment included 1.5 kb total coding region. The recombinant eukaryotic cell expression vector of pcDNA3.1-BACE was constructed successfully, and the sequence of insert was identical to the published sequence. The COS-7 cells transfected with the pcDNA3.1BACE plasmid expressed a high level of the BACE protein in the cytoplasm. Conclusion The recombinant plasmid pcDNA3.1-BACE can provide a very useful tool for the research on the cause of Alzheimer’s disease and lay an important foundation for preventing Alzheimer’s disease.
目的:分析原发性呼吸系统淀粉样变的临床特点,诊断和治疗,并结合文献复习,以提高对该病的诊治水平。方法:回顾性分析我院1998年11月到2008年10月34例原发性呼吸系统淀粉样变性患者,其中发生于咽的淀粉样变性有2例,喉的有27例,气管支气管的有5例.结果:24例行全麻下CO2激光手术治疗,其中3例由于严重的呼吸困难先行支气管切开后再行手术治疗,2例行纤维支气管镜下单纯肿物切除术,余8例由于无法手术而给予对症治疗。结论:呼吸系统淀粉样变性的临床表现缺乏特异性,确诊主要靠病理活检和刚果红染色。目前尚无特异的治疗方法。
Objective To determine the influence and significance of combinative assessment of 64 multi-slice spiral computed tomography (MSCT) with serum amyloid A protein (SAA) or C-reactive protein (CRP) on the selection of operative procedures of rectal cancer under the multi-disciplinary team. Methods Prospectively enrolled patients diagnosed definitely as rectal cancer at West China Hospital of Sichuan University from February to August 2009 were randomly assigned into two groups. In one group named MSCT+SAA group, both 64 MSCT and SAA combinative assessment were made for the preoperative evaluation. In another group named MSCT+CRP group, both MSCT and CRP combinative assessment were made for preoperative evaluation. Furthermore, the preoperative staging and predicted operation procedures were compared with postoperative pathologic staging and practical operation procedures, respectively, and the relationship between the choice of operation procedures and clinicopathologic factors was analyzed. Results All 165 patients were randomly assigned into MSCT+SAA group (n=83) and MSCT+CRP group (n=82). The baseline characteristics of two groups were statistically similar (Pgt;0.05). For MSCT+SAA group, the accuracies of preoperative staging T, N, M and TNM were 74.7%， 68.7%, 100% and 66.3%, respectively. For MSCT+CRP group, the accuracies of preoperative staging T, N, M and TNM were 72.0%, 86.6%, 100% and 81.7%, respectively. There were statistically significant differences in the accuracies of N staging and TNM staging between two groups (P＜0.05). However, there was no statistically significant difference of the accuracy of prediction to operative procedures between two groups (90.4% vs. 95.1%, Pgt;0.05). The pathological T staging (P＜0.001), N staging (P＜0.001), TNM staging (P＜0.001), preoperative serum level of SAA (P=0.010), serum level of CRP (P=0.042), and distance of tumor to the dentate line (P=0.011) were associated with the operative procedures. Conclusion Combinative assessment of MSCT+CRP could improve the accuracy of preoperative staging and operative procedures prediction, which may be superior to MSCT+SAA.